Monoclonal antibody to a human thymocyte antigen and methods of preparing same

ABSTRACT

Hybrid cell line for production of monoclonal antibody to an antigen found on approximately 70% of normal human thymocytes. The hybrid is formed by fusing splenocytes from immunized CAF1 mice with P3X63Ag8U1 myeloma cells. Diagnostic and therapeutic uses of the monoclonal antibody are also disclosed.

FIELD OF THE INVENTION

This invention relates generally to new hybrid cell lines and morespecifically to hybrid cell lines for production of monoclonal antibodyto an antigen found on approximately 70% of normal human thymocytes, tothe antibody so produced, and to therapeutic and diagnositc methods andcompositions employing this antibody.

DESCRIPTION OF THE PRIOR ART

The fusion of mouse myeloma cells to spleen cells from immunized mice byKohler and Milstein in 1975 [Nature 256, 495-497 (1975)] demonstratedfor the first time that it was possible to obtain a continuous cell linemaking homogeneous (so-called "monoclonal") antibody. Since this seminalwork, much effort has been directed to the production of various hybridcells (called "hybridomas") and to the use of the antibody made by thesehybridomas for various scientific investigations. See, for example,Current Topics in Microbiology and Immunology, Volume 81 - "LymphocyteHybridomas", F. Melchers, M. Potter, and N. Warner, Editors,Springer-Verlag, 1978, and references contained therein; C. J.Barnstable, et al., Cell, 14, 9-20 (May, 1978); P. Parham and W. F.Bodmer, Nature 276, 397-399 (November, 1978); Handbook of ExperimentalImmunology, Third Edition, Volume 2, D. M. Wier, Editor, Blackwell,1978, Chapter 25; and Chemical and Engineering News, Jan. 1, 1979,15-17. These references simultaneously indicate the rewards andcomplications of attempting to produce monoclonal antibody fromhybridomas. While the general technique is well understood conceptually,there are many difficulties met and variations required for eachspecific case. In fact, there is no assurance, prior to attempting toprepare a given hybridoma, that the desired hybridoma will be obtained,that it will produce antibody if obtained, or that the antibody soproduced will have the desired specificity. The degree of success isinfluenced principally by the type of antigen employed and the selectiontechnique used for isolating the desired hybridoma.

The attempted production of monoclonal antibody to human lymphocytecell-surface antigens has been reported only in a few instances. See,for example, Current Topics in Microbiology and Immunology, ibid, 66-69and 164-169. The antigens used in these reported experiments werecultured human lymphoblastoid leukemia and human chronic lymphocyticleukemia cell lines. Many hybridomas obtained appeared to produceantibody to various antigens on all human cells. None of the hybridomasproduced antibody against a predefined class of human lymphocytes.

More recently, the present applicants and others have authored articlesdisclosing the preparation and testing of hybridomas making antibody tocertain T-cell antigens. See, for example, Reinherz, E. L., et al., J.Immunol. 123, 1312-1317 (1979); Reinherz, E. L., et al., Proc. Natl.Acad. Sci., 76, 4061-4065 (1979); and Kung, P. C., et al., Science, 206,347-349 (1979).

It should be understood that there are two principal classes oflymphocytes involved in the immune system of humans and animals. Thefirst of these (the thymus-derived cell or T cell) is differentiated inthe thymus from haemopoietic stem cells. While within the thymus, thedifferentiating cells are termed "thymocytes." The mature T cells emergefrom the thymus and circulate between the tissues, lymphatics, and thebloodstream. These T cells form a large proportion of the pool ofrecirculating small lymphocytes. They have immunological specificity andare directly involved in cell-mediated immune responses (such as graftrejection) as effector cells. Although T cells do not secrete humoralantibodies, they are sometimes required for the secretion of theseantibodies by the second class of lymphocytes discussed below. Sometypes of T cells play a regulating function in other aspects of theimmune system. The mechanism of this process of cell cooperation is notyet completely understood.

The second class of lymphocytes (the bone marrow-derived cells or Bcells) are those which secrete antibody. They also develop fromhaemopoietic stem cells, but their differentiation is not determined bythe thymus. In birds, they are differentiated in an organ analogous tothe thymus, called the Bursa of Fabricius. In mammals, however, noequivalent organ has been discovered, and it is thought that these Bcells differentiate within the bone marrow.

It is now recognized that T cells are divided into at least severalsubtypes, termed "helper", "suppressor", and "killer" T cells, whichhave the function of (respectively) promoting a reaction, suppressing areaction, or killing (lysing) foreign cells. These subclasses are wellunderstood for murine systems, but they have only recently beendescribed for human systems. See, for example, R. L. Evans, et al.,Journal of Experimental Medicine, Volume 145, 221-232, 1977; and L.Chess and S. F. Schlossman - "Functional Analysis of Distinct HumanT-Cell Subsets Bearing Unique Differentiation Antigens", in"Contemporary Topics in Immunobiology", O. Stutman, Editor, PlenumPress, 1977, Volume 7, 363-379.

The ability to identify or suppress classes or subclasses of T cells isimportant for diagnosis or treatment of various immunoregulatorydisorders or conditions.

For example, certain leukemias and lymphomas have differing prognosisdepending on whether they are of B cell or T cell origin. Thus,evaluation of the disease prognosis depends upon distinguishing betweenthese two classes of lymphocytes. See, for example, A. C. Aisenberg andJ. C. Long, The American Journal of Medicine, 58:300 (March, 1975); D.Belpomme, et al., in "Immunological Diagnosis of Leukemias andLymphomas", S. Thierfelder, et al., eds, Springer, Heidelberg, 1977,33-45; and D. Belpomme, et al., British Journal of Haematology, 1978,38, 85.

Certain disease states (e.g., juvenile rheumatoid arthritis,malignancies, and agammaglobulinemia) are associated with an imbalanceof T cell subclasses. It has been suggested that autoimmune diseasesgenerally are associated with an excess of "helper" T cells or adeficiency of certain "suppressor" T cells, while agammaglobulinemia isassociated with an excess of certain "suppressor" T cells or adeficiency of "helper" T cells. Malignancies generally are associatedwith an excess of "suppressor" T cells.

In certain leukemias, excess T cells are produced in an arrested stageof development. Diagnosis may thus depend on the ability to detect thisimbalance or excess and to determine which developmental stage is inexcess. See, for example, J. Kersey, et al., "Surface Markers DefineHuman Lymphoid Malignancies with Differing Prognoses" in Haematology andBlood Transfusion, Volume 20, Springer-Verlag, 1977, 17-24, andreferences contained therein; and E. L. Reinherz, et al., J. Clin.Invest., 64, 392-397 (1979).

Acquired agammaglobulinemia, a disease state in which no immune globulinis produced, comprises at least two distinct types. In type I thefailure to produce immune globulin is due to an excess of suppressor Tcells, while in type II it is due to a lack of helper T cells. In bothtypes, there appears to be no defect or lack in the patients' B cells,the lymphocytes which are responsible for the actual secretion of theantibody; however, these B cells are being either suppressed or "nothelped", resulting in greatly decreased or absent immune globulinproduction. The type of acquired agammaglobulinemia may thus bedetermined by testing for an excess of suppressor T cells or an absenceof helper T cells.

On the therapeutic side, there is some suggestion, as yet not definitelyproven, that administration of antibodies against the subtype of T cellin excess may have therapeutic benefit in autoimmune disease ormalignancies. For example, a helper T cell cancer (certain cutaneous Tcell lymphomas and certain T cell acute lymphoblastic leukemias) may betreated by an antibody to a helper T cell antigen. Treatment ofautoimmune disease caused by an excess of helper cells may also beaccomplished in the same fashion. Treatment of diseases (e.g.,malignancies or type I acquired agammaglobulinemia) due to an excess ofsuppressor T cells may be treated by administration of an antibody to asuppressor T cell antigen.

Antisera against the entire class of human T cells (so-called antihumanthymocyte globulin or ATG) has been reported useful therapeutically inpatients receiving organ transplants. Since the cell-mediated immuneresponse (the mechanism whereby transplants are rejected) depends upon Tcells, administration of antibody to T cells prevents or retards thisrejection process. See, for example, Cosimi, et al., "RandomizedClinical Trial of ATG in Cadaver Renal Allgraft Recipients: Importanceof T Cell Monitoring", Surgery 40:155-163 (1976) and referencescontained therein.

The identification and suppression of human T cell classes andsubclasses has previously been accomplished by the use of spontaneousautoantibodies or selective antisera for human T cells obtained byimmunizing animals with human T cells, bleeding the animals to obtainserum, and adsorbing the antiserum with (for example) autologous but notallogeneic B cells to remove antibodies with unwanted reactivities. Thepreparation of these antisera is extremely difficult, particularly inthe adsorption and purification steps. Even the adsorbed and purifiedantisera contain many impurities in addition to the desired antibody,for several reasons. First, the serum contains millions of antibodymolecules even before the T cell immunization. Second, the immunizationcauses production of antibodies against a variety of antigens found onall human T cells injected. There is no selective production of antibodyagainst a single antigen. Third, the titer of specific antibody obtainedby such methods is usually quite low, (e.g., inactive at dilutionsgreater than 1:100) and the ratio of specific to non-specific antibodyis less than 1/10⁶.

See, for example, the Chess and Schlossman article referred to above (atpages 365 and following) and the Chemical and Engineering News articlereferred to above, where the deficiencies of prior art antisera and theadvantages of monoclonal antibody are described.

SUMMARY OF INVENTION

There has now been discovered a novel hybridoma (designated OKT6) whichis capable of producing novel monoclonal antibody against an antigenfound on approximately 70% of normal human thymocytes but not on normalhuman peripheral lymphoid cells (T cells, B cells, or null cells) orbone marrow cells.

The antibody so produced is monospecific for a single determinant onapproximately 70% of normal human thymocytes and contains essentially noother anti-human immune globulin, in contrast to prior art antisera(which are inherently contaminated with antibody reactive to numeroushuman antigens) and to prior art monoclonal antibodies (which are notmonospecific for a human thymocyte antigen). Moreover, this hybridomacan be cultured to produce antibody without the necessity of immunizingand killing animals, followed by the tedious adsorption and purificationsteps necessary to obtain even the impure antisera of the prior art.

It is accordingly one object of this invention to provide hybridomaswhich produce antibodies against an antigen found on about 70% of normalhuman thymocytes.

It is a further aspect of the present invention to provide methods forpreparing these hybridomas.

A further object of the invention is to provide essentially homogeneousantibody against an antigen found on about 70% of normal humanthymocytes.

A still further object is to provide methods for treatment or diagnosisof disease or for identification of T cell or thymocyte subclassesemploying this antibody.

Other objects and advantages of the invention will become apparent fromthe examination of the present disclosure.

In satisfaction of the foregoing objects and advantages, there isprovided by this invention a novel hybridoma producing novel antibody toan antigen found on approximately 70% of normal human thymocytes (butnot on normal human peripheral lymphoid cells or bone marrow cells), theantibody itself, and diagnostic and therapeutic methods employing theantibody. The hybridoma was prepared generally following the method ofMilstein and Kohler. Following immunization of mice with normal humanthymocytes, the spleen cells of the immunized mice were fused with cellsfrom a mouse myeloma line and the resultant hybridomas screened forthose with supernatants containing antibody which gave selective bindingto normal E rosette positive human T cells and/or thymocytes. Thedesired hybridomas were subsequently cloned and characterized. As aresult, a hybridoma was obtained which produces antibody (designatedOKT6) against an antigen on approximately 70% of normal humanthymocytes. Not only does this antibody react with about 70% of normalhuman thymocytes, but it also does not react with normal peripheralblood lymphoid cells or bone marrow cells.

In view of the difficulties indicated in the prior art and the lack ofsuccess reported using malignant cell lines as the antigen, it wassurprising that the present method provided the desired hybridoma. Itshould be emphasized that the unpredictable nature of hybrid cellpreparation does not allow one to extrapolate from one antigen or cellsystem to another. In fact, the present applicants have discovered thatthe use of a T cell malignant cell line or purified antigens separatedfrom the cell surface as the antigen were generally unsuccessful.

Both the subject hybridoma and the antibody produced thereby areidentified herein by the designation "OKT6", the particular materialreferred to being apparent from the context. The subject hybridoma wasdeposited on Nov. 21, 1979 at the American Type Culture Collection,12301 Parklawn Drive, Rockville, Maryland 20852, and was given the ATCCaccession number CRL 8020.

The preparation and characterization of the hybridoma and the resultantantibody will be better understood by reference to the followingdescription and Examples.

DETAILED DESCRIPTION OF THE INVENTION

The method of preparing the hybridoma generally comprises the followingsteps:

A. Immunizing mice with normal human thymocytes. While it has been foundthat female CAF₁ mice are preferred, it is contemplated that other mousestrains could be used. The immunization schedule and thymocyteconcentration should be such as to produce useful quantities of suitablyprimed splenocytes. Three immunizations at fourteen day intervals with2×10⁷ cells/mouse/injection in 0.2 ml phosphate buffered saline has beenfound to be effective.

B. Removing the spleens from the immunized mice and making a spleensuspension in an appropriate medium. About one ml of medium per spleenis sufficient. These experimental techniques are well-known.

C. Fusing the suspended spleen cells with mouse myeloma cells from asuitable cell line by the use of a suitable fusion promoter. Thepreferred ratio is about 5 spleen cells per myeloma cell. A total volumeof about 0.5-1.0 ml of fusion medium is appropriate for about 10⁸splenocytes. Many mouse myeloma cell lines are known and available,generally from members of the academic community or various depositbanks, such as the Salk Institute Cell Distribution Center, La Jolla,CA. The cell line used should preferably be of the so-called "drugresistant" type, so that unfused myeloma cells will not survive in aselective medium, while hybrids will survive. The most common class is8-azaguanine resistant cell lines, which lack the enzyme hypoxanthineguanine phophoribosyl transferase and hence will not be supported by HAT(hypoxanthine, aminopterin, and thymidine) medium. It is also generallypreferred that the myeloma cell line used be of the so-called"non-secreting" type, in that it does not itself produce any antibody,although secreting types may be used. In certain cases, however,secreting myeloma lines may be preferred. While the preferred fusionpromoter is polyethylene glycol having an average molecular weight fromabout 1000 to about 4000 (commercially available as PEG 1000, etc.),other fusion promoters known in the art may be employed.

D. Diluting and culturing in separate containers, the mixture of unfusedspleen cells, unfused myeloma cells, and fused cells in a selectivemedium which will not support the unfused myeloma cells for a timesufficient to allow death of the unfused cells (about one week). Thedilution may be a type of limiting one, in which the volume of diluentis statistically calculated to isolate a certain number of cells (e.g.,1-4) in each separate container (e.g., each well of a microtiter plate).The medium is one (e.g., HAT medium) which will not support the drugresistant (e.g., 8-azaguanine resistant) unfused myeloma cell line.Hence, these myeloma cells perish. Since the unfused spleen cells arenon-malignant, they have only a finite number of generations. Thus,after a certain period of time (about one week) these unfused spleencells fail to reproduce. The fused cells, on the other hand, continue toreproduce because they possess the malignant quality of the myelomaparent and the ability to survive in the selective medium of the spleencell parent.

E. Evaluating the supernatant in each container (well) containing ahybridoma for the presence of antibody to E rosette positive purifiedhuman T cells or thymocytes.

F. Selecting (e.g., by limiting dilution) and cloning hybridomasproducing the desired antibody.

Once the desired hybridoma has been selected and cloned, the resultantantibody may be produced in one of two ways. The purest monoclonalantibody is produced by in vitro culturing of the desired hybridoma in asuitable medium for a suitable length of time, followed by recovery ofthe desired antibody from the supernatant. The suitable medium andsuitable length of culturing time are known or are readily determined.This in vitro technique produces essentially monospecific monoclonalantibody, essentially free from other specific antihuman immuneglobulin. There is a small amount of other immune globulin present sincethe medium contains xenogeneic serum (e.g., fetal calf serum). However,this in vitro method may not produce a sufficient quantity orconcentration of antibody for some purposes, since the concentration ofmonoclonal antibody is only about 50 μg/ml.

To produce a much greater concentration of slightly less pure monoclonalantibody, the desired hybridoma may be injected into mice, preferablysyngenic or semi-syngenic mice. The hybridoma will cause formation ofantibody-producing tumors after a suitable incubation time, which willresult in a high concentration of the desired antibody (about 5-20mg/ml) in the bloodstream and peritoneal exudate (ascites) of the hostmouse. Although these host mice also have normal antibodies in theirblood and ascites, the concentration of these normal antibodies is onlyabout 5% of the monoclonal antibody concentration. Moreover, since thesenormal antibodies are not antihuman in their specificity, the monoclonalantibody obtained from the harvested ascites or from the serum isessentially free of any contaminating antihuman immune globulin. Thismonoclonal antibody is high titer (active at dilutions of 1:50,000 orhigher) and high ratio of specific to non-specific immune globulin(about 1/20). Immune globulin produced incorporating the light myelomachains are non-specific, "nonsense" peptides which merely dilute themonoclonal antibody without detracting from its specificity.

EXAMPLE I Production of Monoclonal Antibodies A. Immunization andSomatic Cell Hybridization

Female CAF₁ mice (Jackson Laboratories; 6-8 weeks old) were immunizedintraperitoneally with 2×10⁷ human thymocytes in 0.2 ml of phosphatebuffered saline at 14-day intervals. Four days after the thirdimmunization, spleens were removed from the mice, and a single cellsuspension was made by pressing the tissue through a stainless steelmesh.

Cell fusion was carried out according to the procedure developed byKohler and Milstein. 1×10⁸ splenocytes were fused in 0.5 ml of a fusionmedium comprising 35% polyethylene glycol (PEG 1000) and 5%dimethylsulfoxide in RPMI 1640 medium (Gibco, Grand Island, NY) with2×10⁷ P3X63Ag8U1 myeloma cells supplied by Dr. M. Scharff, AlbertEinstein College of Medicine, Bronx, NY. These myeloma cells secreteIgG₁ K light chains.

B. Selection and Growth of Hybridoma

After cell fusion, cells were cultured in HAT medium (hypoxanthine,aminopterin, and thymidine) at 37° C. with 5% CO₂ in a humid atmosphere.Several weeks later, 40 to 100 μl of supernatant from culturescontaining hybridomas were added to a pellet of 10⁶ peripherallymphocytes separated into E rosette positive (E⁺) and E rosettenegative (E⁻) populations, which were prepared from blood of healthyhuman donors as described by Mendes (J. Immunol. 111:860, 1973).Detection of mouse hybridoma antibodies binding to these cells wasdetermined by indirect immunofluorescence. Cells incubated with culturesupernatants were stained with a fluorescinated goat-anti-mouse IgG (G/MFITC) (Meloy Laboratories, Springfield, VA; F/p=2.5) and the fluorescentantibody-coated cells were subsequently analyzed on the CytofluorografFC200/4800A (Ortho Instruments, Westwood, MA) as described in ExampleIII. Hybridoma cultures containing antibodies reacting specifically withE⁺ lymphocytes (T cells) and/or thymocytes were selected and clonedtwice by limiting dilution methods in the presence of feeder cells.Subsequently, the clones were transferred intraperitoneally by injecting1×10.sup. 7 cells of a given clone (0.2 ml volume) into CAF₁ mice primedwith 2,6,10,14-tetramethylpentadecane, sold by Aldrich Chemical Companyunder the name Pristine. The malignant ascites from these mice were thenused to characterize lymphocytes as described below in Example II. Thesubject hybrid antibody OKT6 was demonstrated by standard techniques tobe of IgG₁ subclass.

EXAMPLE II Characterization of OKT6 Reactivity A. Isolation ofLymphocyte Populations

Human peripheral blood mononuclear cells were isolated from healthyvolunteer donors (ages 15-40) by Ficoll-Hypaque density gradientcentrifugation (Pharmacia Fine Chemicals, Piscataway, NJ) following thetechnique of Boyum, Scand. J. Clin. Lab. Invest. 21 (Suppl. 97): 77,1968. Unfractionated mononuclear cells were separated into surface Ig⁺(B) and Ig⁻ (T plus Null) populations by Sephadex G-200 anti-F(ab')₂column chromatography as previously described by Chess, et al., J.Immunol. 113:1113 (1974). T cells were recovered by E rosetting the Ig⁻population with 5% sheep erythrocytes (microbiological Associates,Bethesda, MD). The rosetted mixture was layered over Ficoll-Hypaque andthe recovered E⁺ pellet treated with 0.155M NH₄ Cl (10 ml per 10⁸cells). The T cell population so obtained was <2% EAC rosette positiveand >95% E rosette positive as determined by Standard methods. Inaddition, the non-rosetting Ig⁻ (Null cell) population was harvestedfrom the Ficoll interface. This latter population was <5% E⁺ and >2%sIg⁺ . The surface Ig⁺ (B) population was obtained from the SephadexG-200 column following elution with normal human gamma globulin aspreviously described. This population was >95% surface Ig⁺ and <5% E⁺.

Normal human bone marrow cells were obtained from the posterior iliaccrest of normal human volunteers by needle aspiration.

B. Isolation of Thymocytes

Normal human thymus gland was obtained from patients aged two months to14 years undergoing corrective cardiac surgery. Freshly obtainedportions of the thymus gland were immediately placed in 5% fetal calfserum in medium 199 (Gibco), finely minced with forceps and scissors,and subsequently made into single cell suspensions by being pressedthrough wire mesh. The cells were next layered over Ficoll-Hypaque andspun and washed as previously described in section A above. Thethymocytes so obtained were >95% viable and ≧90% E rosette positive.

C. Cell Lines of T Lineage and T Acute Lymphoblastic Leukemia Cells

T cell lines CEM and HSB-2, and MOLT-4 were provided by Dr. H. Lazarus(Sidney Farber Cancer Institute, Boston, MA). Leukemic cells wereobtained from 25 patients with the diagnosis of T cell ALL. Theseindividual tumors had been previously determined to be of T cell lineageby their spontaneous rosette formation with sheep erythrocytes (>20%E⁺), and reactivity with T cell specific heteroantisera anti-HTL (B.K.)and A99, as previously described. Tumor populations were cryopreservedat -196° C. vapor phase liquid nitrogen with 10% DMSO and 20% AB humanserum until the time of surface characterization. All tumor populationsanalyzed were more than 90% blasts by Wright-Giemsa morphology ofcytocentrifuge preparations.

EXAMPLE III Cytofluorographic Analysis and Cell Separation

Cytofluorographic analysis of monoclonal antibodies with all cellpopulations was performed by indirect immunofluorescence withfluorescein-conjugated goat anti-mouse IgG (G/M FITC) (MeloyLaboratories) utilizing a Cytofluorograf FC200/4800A (OrthoInstruments). In brief, 1×10⁶ cells were treated with 0.15 ml OKT5 at a1:500 dilution, incubated at 4° C. for 30 minutes, and washed twice. Thecells were then reacted with 0.15 ml of a 1:40 dilution G/M FITC at 4°C. for 30 minutes, centrifuged, and washed three times. Cells were thenanalyzed on the Cytofluorograf, and the intensity of fluorescence percell was recorded on a pulse height analyzer. A similar pattern ofreactivity was seen at a dilution of 1:10,000, but further dilutioncaused loss of reactivity. Background staining was obtained bysubstituting a 0.15 ml aliquot of 1:500 ascites from a CAF₁ mouseintraperitoneally injected with a non-producing hybrid clone.

In experiments involving antibody and complement mediated lympholysis,thymocytes and peripheral T cells were cultured overnight followingselective lysis and then subsequently analyzed on the Cytofluorograf.

EXAMPLE IV Lysis of Lymphoid Populations with Monoclonal Antibody andComplement

Forty×10⁶ peripheral T cells or thymocytes were placed in a 15 mlplastic tube (Falcon, Oxnard, CA). Cell pellets were incubated with 0.8cc of OKT3, OKT4, OKT8, or normal ascites control diluted 1:200 in PBS,resuspended, and incubated at 20° C. for 60 minutes. Subsequently, 0.2cc of fresh rabbit complement was added to the antibody treatedpopulations, resuspended, and further incubated at 37° C. in a shakingwater bath for 60 minutes. At the end of this time, cells were spun downand viable cells enumerated by Trypan blue exclusion. After counting,cells were washed two additional times in 5% FCS and placed in finalmedia [RPMI 1640 (Grand Island Biological Company, Grand Island, NY)containing 20% AB⁺ human serum, 1% penicillin-streptomycin, 200 mML-glutamine, 25 mM HEPES buffer, and 0.5% sodium bicarbonate] andincubated overnight in a humid atmosphere with 5% CO₂ at 37° C.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the fluorescence pattern obtained on the Cytofluorografafter reacting normal human thymocytes with OKT6 and other monoclonalantibodies at a 1:500 dilution and G/M FITC. Background fluorescencestaining was obtained by incubating each population with a 1:500dilution of ascitic fluid from a mouse injected with a non-producingclone.

FIG. 2 shows the stages of intrathymic differentiation in man.

The production of the hybridoma and the production and characterizationof the resulting monoclonal antibody were conducted as described in theabove Examples. Although large quantities of the subject antibody wereprepared by injecting the subject hybridoma intraperitoneally into miceand harvesting the malignant ascites, it is clearly contemplated thatthe hybridoma could be cultured in vitro by techniques well-known in theart and the antibody removed from the supernatant.

Table 1 shows the reactivity of OKT6, OKT8, OKT9, and OKT10 with varioushuman lymphoid cell populations. The OKT6 monoclonal antibody isreactive with approximately 70% of normal human thymocytes and not withany other lymphoid cells tested. This pattern of reactivity is one testby which the subject antibody OKT6 may be detected and distinguishedfrom other antibodies.

FIG. 1 shows a representative fluorescense pattern obtained on theCytofluorograf after reacting normal human thymocyte suspensions with a1:500 dilution of OKT3, OKT4, OKT5, OKT6, OKT8, OKT9, OKT10, and G/MFITC. Similar patterns of reactivity were seen with 12 additional normalhuman thymocyte populations tested. As shown, significant differencesexist in both the percentage of reactivity and fluorescence intensitywith each of these monoclonal antibodies. For example, OKT9 reacts withapproximately 10% of thymocytes with low fluorescence intensity whileOKT5, OKT6, OKT8 and OKT10 react with approximately 70% of thymocytes ata higher fluorescence intensity. OKT4, which reacts with 75% ofthymocytes, is intermediate between OKT9 and the monoclonal antibodieswhich give a pattern of greater fluorescence intensity. In addition,FIG. 1 shows that approximately 15% of thymocytes are detected with OKT3by indirect immunofluorescence. Not shown is OKT1, whose pattern ofreactivity is virtually identical to OKT3 on thymocytes. The pattern ofreactivity in FIG. 1 is another test by which the subject antibody OKT6may be detected and distinguished from other antibodies.

Table 2 shows the distribution of antigens defined by various monoclonalantibodies on human peripheral T cells and lymphocytes, as determined bythe series of lysis experiments described in Example IV. Since onlyOKT3, OKT4, and OKT8 were complement fixing monoclonal antibodies, thesethree were utilized.

As shown in Table 2A, the entire T cell population reacts with OKT3while OKT4, OKT5, and OKT8 react with 60%, 25%, and 34% of T cells,respectively. Lysis with OKT4 and complement diminished the total numberby 62% and specifically deleted the OKT4⁺ population. In addition, thepercentage of OKT5⁺ and OKT8⁺ cells increased and there was no effect onthe absolute number of OKT5⁺ and OKT8⁺ T cells. These experimentssuggested that OKT4⁺ was distinct from the OKT5⁺ and OKT8⁺ populations.Further support for this conclusion was obtained by lysis of T cellswith OKT8 and complement. In this case, the percentage of OKT4⁺ T cellsincreased, the absolute number remained the same, and OKT8⁺ and OKT5⁺populations were eliminated. Moreover, these results demonstrated thatthe OKT8⁺ population was reciprocal to the OKT4⁺ population andcontained the entire OKT5⁺ T cell subset.

Similar experiments with human thymocyte populations gave differentresults. As shown in Table 2B, approximately 75% of thymocytes wereOKT4⁺ or OKT8⁺. Moreover, following lysis with either OKT4 or OKT8, only25% of thymocytes remained. The majority of residual thymocytes werereactive with OKT3, whereas only a minority was reactive with OKT6.These findings demonstrate that a major population of human thymocytesbear the OKT4, OKT5, OKT6, and OKT8 surface antigens on the same cell.In addition, Table 2 demonstrates that following treatment with OKT8 orOKT4, there is a marked increase in the mature thymocytes bearing theOKT3 antigen. Thus, the majority of OKT3 reactive thymocytes havealready segregated into OKT4⁺ or OKT8⁺ subsets, since the majorproportion of residual cells following OKT4 or OKT8 lysis are OKT3⁺. Ifthe OKT3⁺ subpopulation were both OKT4⁺ and OKT8⁺, then lysis witheither monoclonal antibody should have removed the OKT3 reactivethymocytes.

To further determine the relationship of OKT3 relative thymocytesubpopulations to the other monoclonal antibody defined thymocytefraction, thymocytes were treated with OKT3 and complement and theresidual cells were then compared to untreated thymocyte populations. Asshown in Table 2B, OKT3 and complement removed 25% of thymocytes.Moreover, there was no major loss of OKT4, OKT5, OKT6, or OKT8 reactivepopulations. These findings suggest that the vast majority of thymocytesbearing the OKT6 marker are contained in the OKT3⁻ population. Inaddition, they further suggest that thymocytes simultaneously expressingantigens defined by OKT4, OKT5, and OKT8 are likewise restricted to theOKT3⁻ population. It should also be noted that the OKT9 reactivepopulation of thymocytes was not diminished following OKT3 andcomplement treatment of the unfractionated thymocytes, thus showing thatthe OKT9⁺ subpopulation is largely restricted to the OKT3⁻ thymocytepopulation.

Based upon these results, it has been possible to describe the stages ofintrathymic development of human thymocytes. As shown in FIG. 2,virtually all thymocytes bear the OKT10 marker. In addition, thymocytesacquire at an early stage the OKT9 marker (Thyl and Thy2, respectively).This stage defines the minority of thymocytes and accounts forapproximately 10% of the unfractionated population. Subsequently, humanthymocytes acquire a thymocyte unique antigen defined by OKT6 andconcurrently express OKT4, OKT5, and OKT8 (Thy4). This lattersubpopulation represents the majority of thymocytes and accounts forupwards of 70-80% of the thymic population. With further maturation,thymocytes lose OKT6 reactivity, acquire OKT3 (and OKT1) reactivity, andsegregate into OKT4⁺ and OKT5⁺ /OKT8⁺ subsets (Thy7 and Thy8). Lastly,it appears that as the thymocyte is exported into the peripheral T cellcompartment, it loses the OKT10 marker since this antigen is lacking onvirtually all peripheral T lymphocytes. Possible transitional statesbetween these three major stages of thymic development are designated byThy3, Thy5, and Thy6 in FIG. 2.

Since acute lymphoblastic leukemia of T lineage is thought to be derivedfrom immature thymocytes, the relationship between tumor cells fromindividuals with T-ALL and these proposed stages of intrathymicdifferentiation was determined. Twenty-five tumor cell populations fromindividuals with T-ALL and three T cell lines previously studied withconventional Anti-T cell reagents and E rosetting were investigated. Asshown in Table 3, the majority of T-ALL leukemic cells were reactivewith either OKT10 alone or OKT9 and OKT10 and failed to react with theother monoclonal antibodies. Thus, 15/25 cases studied appeared topossess early thymocyte antigens (Stage I).

In contrast, 5/25 cases were reactive with OKT6, suggesting derivationfrom a more mature thymus population (Stage II). This T-ALL Group wasitself heterogeneous with respect to OKT4, OKT8, and OKT9 reactivity asshown in Table 3. Cells from 2/5 patients possess most of the commonthymocyte antigens including OKT4, OKT6, and OKT8. It is worthy of notethat OKT5 is not present on any of these 5 Stage II tumors even thoughOKT8 reactivity was observed. This latter result clearly suggests thatOKT5 and OKT8 define different antigens or different determinants on thesame antigen. Finally, 1/25 patients' tumors came from a maturethymocyte population (Stage III) as defined by its reactivity with OKT3.This individual's tumor, in addition, was reactive with OKT5, OKT8, andOKT10. Of the 25 leukemic populations analyzed, only four tumors couldnot be clearly categorized. Three were positive with OKT4 and OKT8, butlacked OKT3 and OKT6 and most likely represented transitions from Thy4and Thy7,8. One of 25 cases appeared to be a transition from Thy3 toThy4 since it possessed OKT8 and OKT10 reactivity.

T cell lines derived from T-ALL tumor populations also represented cellsfrom a specific state of intrathymic differentiation. As shown in Table4, HSB was reactive with OKT9 and OKT10 exclusively and would thereforedefine a tumor population derived from Stage I. In contrast, CEM wasreactive with OKT4, OKT6, OKT8, OKT9, and OKT10 and appeared to derivefrom a Stage II thymocyte. Finally, MOLT-4 seems to represent a leukemictransformation at a stage between HSB-2 and CEM since it expressed OKT6,OKT8, OKT9, and OKT10.

Since patients with later stages (e.g., Stage II) of T cell acutelymphoblastic leukemia have been shown to have more prolongeddisease-free survival than those with Stage I ALL, the use of OKT6antibody allows conclusions concerning the prognosis of a given patientwith T-cell ALL.

Table 5 shows the relationship between levels of peripheral T cells andT cell subsets and various disease states. These relationships may beused for diagnostic purposes (e.g., to detect acute infectiousmononucleosis) by analyzing the blood sample of an individual suspectedof having one of these disease states to determine the levels of T cellsand T cell subsets. These relationships may also be used for therapeuticpurposes where the cause of the disease state is an elevated level of aT cell subset (e.g., Type I acquired agammaglobulinemia). Fortherapeutic use, administration of the appropriate monoclonal antibodyto a patient with an elevated T cell subset level will decrease oreliminate the excess. The relationships shown in Tables 2-5 are afurther way in which OKT6 antibody may be detected and distinguishedfrom other antibodies.

Other monoclonal antibody producing hybridomas prepared by the presentapplicants (designated OKT1, OKT3, OKT4, and OKT5) are described andclaimed in the following U.S. patent applications: Ser. Nos. 22,132,filed Mar. 20, 1979; 33,639, filed Apr. 26, 1979; 33,669, filed Apr. 26,1979; and 76,642, filed Sept. 18, 1979; and 82,515, filed Oct. 9, 1979.Still other monoclonal antibody producing hybridomas prepared by thepresent applicants (designated OKT8, OKT9, and OKT10) are described andclaimed in U.S. patent applications filed on even date herewith andentitled:

Hybrid Cell Line for Producing Complement-Fixing Monoclonal Antibody toHuman Suppressor T Cells, Antibody, and Methods; Hybrid Cell Line ForProducing Monoclonal Antibody to Human Early Thymocyte Antigen,Antibody, and Methods; and Hybrid Cell Line For Producing MonoclonalAntibody to a Human Prothymocyte Antigen, Antibody, and Methods

These applications are incorporated herein by reference.

The hybridomas disclosed are claimed in the above patent applicationshave also been deposited at the American Type Culture Collection andassigned the following designations: OKT 1 - CRL 8000; OKT 3 - CRL 8001;OKT 4 -CRL 8002; OKT 5 - CRL 8013; OKT 6 - CRL 8020; OKT 8 - CRL 8014;OKT 9 - CRL 8021; OKT 10 - 8022.

According to the present invention there are provided a hybridomacapable of producing antibody against an antigen found on approximately70% of normal human thymocytes, a method for producing this hybridoma,monoclonal antibody against an antigen found on approximately 70% ofnormal human thymocytes, methods for producing the antibody, and methodsand compositions for treatment or diagnosis of disease or identificationof T cell or thymocyte subclasses employing this antibody.

                  TABLE 1                                                         ______________________________________                                        REACTIVITY OF MONOCLONAL ANTIBODIES ON                                        HUMAN LYMPHOID POPULATIONS                                                             Peripheral                                                           Monoclonal                                                                             Blood (30)*  Bone                                                    Antibody E.sup.+  E.sup.- Marrow (6)                                                                            Thymus (22)                                 ______________________________________                                        OKT6     0%       0%      0%      70%                                         OKT8     30%      0%      <2%     80%                                         OKT9     0%       0%      0%      ≦10%                                  OKT10   <5%      10%     ≦20%                                                                           95%                                         ______________________________________                                         *Numbers in parentheses represent the number of samples tested; % values      are means.                                                               

                                      TABLE 2                                     __________________________________________________________________________    DIFFERENCES IN DISTRIBUTION OF ANTIGENS DEFINED BY                            MONOCLONAL ANTIBODY ON HUMAN PERIPHERAL T CELLS AND THYMOCYTES                                      Percent Reactivity of Residual Cells With               Lymphoid    Total Cell                                                                              Monoclonal Antibodies:                                  Population  Number Recovered                                                                        OKT3                                                                              OKT4                                                                              OKT5                                                                              OKT6                                                                              OKT8                                                                              OKT9                                                                              OKT10                           __________________________________________________________________________    A.                                                                              Peripheral T Cells                                                            Untreated*                                                                                40 × 10.sup.6                                                                   98  60  25   0  34  --  --                                OKT4 + C'.sup.+                                                                         15.2 × 10.sup.6                                                                   95   0  70   0  95  --  --                                OKT8 + C' 25.2 × 10.sup.6                                                                   95  92   0   0   0  --  --                              B.                                                                              Thymocytes                                                                    Untreated*                                                                              40 × 10.sup.6                                                                     30  75  70  65  75  8   92                                OKT4 + C' 10 × 10.sup.6                                                                     80   0  85  10  55  6   75                                OKT8 + C' 9.5 × 10.sup.6                                                                    85  65   0  13   0  5   85                                OKT3 + C' 30 × 10.sup.6                                                                      0  60  80  82  90  12  86                              __________________________________________________________________________     *Untreated populations and populations treated with complement alone were     indistinguishable on reanalysis. Nonspecific lysis was ≦5% in all      cases. Results are representative of 6 experiments.                           .sup.+ C' = complement                                                   

                                      TABLE 3                                     __________________________________________________________________________    CELL SURFACE CHARACTERISTICS OF ACUTE LYMPHOBLASTIC                           LEUKEMIA OF T-LINEAGE                                                         Differentiative                                                                             Reactivity with Monoclonal Antibodies:                                                                     Number of T-ALL Tested               Status      OKT3                                                                              OKT4                                                                              OKT5                                                                              OKT6                                                                              OKT8                                                                              OKT9                                                                              OKT10                                                                              (n = 25)                           __________________________________________________________________________    Stage I                                                                       A. Prothymocyte (Thyl).sup.+                                                                -   -   -   -   -   -     +.sup.++                                                                         7                                  B. Early thymocyte (Thy2)                                                                   -   -   -   -   -   +   +    8                                  Stage II                                                                      Common thymocyte                                                              (Thy3)        -   +   -   +   +   +   +    1                                  (Thy4)        -   +   -   +   +   -   +    1                                  (Thy3-4)      -   -   -   +   -   -   +    2                                  (Thy4-6)      -   -   -   +   +   -   +    1                                  Stage III                                                                     Late thymocyte (Thy8)                                                                       +   -   +   -   +   -   +    1                                                                             21*                                __________________________________________________________________________     *An additional four tumors could not be easily categorized into Stage         I-III. See text for details of their characterization.                        .sup.+ Thy designation refers to FIG.                                         .sup.++ Positive (+) reactivity was defined as ≧30% specific           fluorescence above background control while negative (-) reactivity was       indistinguishable from background staining on tumor cell suspensions.    

                                      TABLE 4                                     __________________________________________________________________________    REACTIVITY WITH MONOCLONAL ANTIBODIES                                         Cell Line                                                                           OKT3 OKT4                                                                              OKT5 OKT6                                                                              OKT8 OKT9                                                                              OKT10                                        __________________________________________________________________________    HSB-2  -*  -   -    -   -    +   +                                            CEM   -    +   -    +   +    +   +                                            MOLT-4                                                                              -    -   -    +   +    +   +                                            __________________________________________________________________________     *Criteria for - and + reactivity was the same as in Table 3.             

                  TABLE 5                                                         ______________________________________                                        PERIPHERAL T CELL LEVELS IN DISEASE STATES                                            T Cell Levels                                                         Disease State                                                                           OKT3.sup.+                                                                             OKT4.sup.+                                                                              OKT5  OKT8  OKT6                                 ______________________________________                                        Primary Biliary                                                                         N        +         -     -     -                                    Cirrhosis (2)                                                                 Multiple  -        N         -     -     -                                    Sclerosis                                                                     (advanced -disease) (8)                                                       Myasthenia                                                                              O        O         O     O     O                                    Gravis (early                                                                 untreated) (3)                                                                Acute Graft vs                                                                          O to -   -         O     +     +                                    Host (3)                                                                      Acquired                                                                      Agamma                                                                        globulinemia                                                                  Type I                       +                                                Type II            O                                                          Hyper IgE (4)                                                                           -        N         O to -                                                                              O to -                                                                              -                                    Acute     +        O to --   ++    ++    O                                    Infectious                                                                    Mononucleosis                                                                 (4)*                                                                          Hodgkins                                                                      Disease                                                                       Stages I & II                                                                           N        N         N     N     O                                    Stages III &                                                                            --       N         N     N     O                                    IV                                                                            Psoriasis (3/5)                                                                         N        + to ++   N     N     O                                    ______________________________________                                         N = within normal limits                                                      O = absent                                                                    + = above normal                                                              ++ = greatly above normal                                                      - = below normal                                                             -- = greatly below normal                                                     *these levels return to normal about one week prior to the disappearance      of clinical symptoms                                                          The numbers in parentheses indicate the number of patients evaluated.    

Although only a single hybridoma producing a single monoclonal antibodyagainst a human thymocyte antigen is described, it is contemplated thatthe present invention encompasses all monoclonal antibodies exhibitingthe characteristics described herein. It was determined that the subjectantibody OKT6 belongs to the subclass IgG₁, which is one of foursubclasses of murine IgG. These subclasses of immune globulin G differfrom one another in the so-called "fixed" regions, although an antibodyto a specific antigen will have a so-called "variable" region which isfunctionally identical regardless of which subclass of immune globulin Git belongs to. That is, a monoclonal antibody exhibiting thecharacteristic described herein may be of subclass IgG₁, IgG₂ a, IgG₂ b,or IgG₃, or of classes IgM, IgA, or other known Ig classes. Thedifferences among these classes or subclasses will not affect theselectivity of the reaction pattern of the antibody, but may affect thefurther reaction of the antibody with other materials, such as (forexample) complement or anti-mouse antibodies. Although the subjectantibody is specifically IgG₁, it is contemplated that antibodies havingthe patterns of reactivity illustrated herein are included within thesubject invention regardless of the immune globulin class or subclass towhich they belong.

Further included within the subject invention are methods for preparingthe monoclonal antibodies described above employing the hybridomatechnique illustrated herein. Although only one example of a hybridomais given herein, it is contemplated that one skilled in the art couldfollow the immunization, fusion, and selection methods provided hereinand obtain other hybridomas capable of producing antibodies having thereactivity characteristics described herein. Since the individualhybridoma produced from a known mouse myeloma cell line and spleen cellsfrom a known species of mouse cannot be further identified except byreference to the antibody produced by the hybridoma, it is contemplatedthat all hybridomas producing antibody having the reactivitycharacteristics described above are included within the subjectinvention, as are methods for making this antibody employing thehybridoma.

Further aspects of the invention are methods of treatment or diagnosisof disease employing the monoclonal antibody OKT6 or any othermonoclonal antibody exhibiting the pattern of reactivity providedherein. The subject antibody may be used to detect and study intrathymicdifferentiation as summarized in FIG. 2. Abnormalities in Stage IIdifferentiation would be indicated by a deviation from about 70% OKT6⁺thymocytes. Moreover, the subject antibody may be employed to diagnosedisease states as shown in Table 5. These techniques may be employedusing OKT6 antibody alone or in combination with other antibodies (e.g.,OKT3-OKT10). Patterns of reactivity with a panel of antibodies to Tcells and T cell subsets will allow more precise detection of certaindisease states then is possible using prior diagnostic methods.

Treatment of disease states (e.g., malignancies such as Stage II ALL)manifesting themselves as an excess of OKT6⁺ cells may be accomplishedby administration of a therapeutically effective amount of OKT6 antibodyto an individual in need of such treatment. By selective reaction withOKT6⁺ antigen, the effective amount of OKT6 antibody will reduce theexcess of OKT6⁺ cells, thus ameliorating the effects of the excess.Diagnostic and therapeutic compositions comprising effective amounts ofOKT6 antibody in admixture with diagnostically or pharmaceuticallyacceptable carriers, respectively, are also included within the presentinvention.

What is claimed is:
 1. A monoclonal antibody of class IgG produced by ahybridoma formed by fusion of cells from a mouse myeloma line and spleencells from a mouse previously immunized with human thymocytes, whichantibody:(a) reacts with approximately 70 percent of normal humanthymocytes, but not with normal human peripheral T cells, B cells, Nullcells, or bone marrow cells, (b) reacts with about 65 percent ofuntreated thymocytes, 10 percent of thymocytes previously treated withmonoclonal antibody produced by hybrid cell line ATCC CRL 8002 andcomplement, 82 percent of thymocytes previously treated with monoclonalantibody produced by hybrid cell line ATCC CRL 8001 and complement, and13 percent of the thymocytes previously treated with monoclonal antibodyproduced by hybrid cell line ATCC CRL 8014 and complement; (c) reactswith stage II T-cell ALL but not with stage I or stage III T-cell ALL;(d) reacts with CEM and MOLT-4 cell lines but not with HSB-2 cell line;and (e) defines a T cell population which is lower than normal levels inprimary biliary cirrhosis, multiple sclerosis, and hyper IgE, higherthan normal levels in acute graft versus host reaction, and completelyabsent in myasthenia gravis, acute infectious mononucleosis, all stagesof Hodgkins disease, and psoriasis.
 2. The monoclonal antibody of claim1 which is of subclass IgG₁.
 3. The monoclonal antibody of claim 1 whichis produced from a hybridoma formed by fusion of P3X63Ag8U1 myelomacells and spleen cells from a CAF₁ mouse previously immunized with humanthymocytes.
 4. A monoclonal antibody of class IgG produced by ahybridoma formed by fusion of cells from a mouse myeloma line and spleencells from a mouse previously immunized with human thymocytes whichreacts with approximately 70 percent of normal human thymocytes, but notwith normal human peripheral T cells, B cells, Null cells, or bonemarrow cells.
 5. Mouse monoclonal antibody which reacts withapproximately 70% of normal human thymocytes but not with normal humanperipheral T cells, B cells, null cells, or bone marrow cells.
 6. Amonoclonal antibody which reacts with approximately 70% of normal humanthymocytes but not with normal human peripheral T cells, B cells, nullcells, or bone marrow cells, prepared by the method which comprises thesteps of:(i) immunizing mice with normal human thymocytes; (ii) removingthe spleens from said mice and making a suspension of the spleen cells;(iii) fusing said spleen cells with mouse myeloma cells in the presenceof a fusion promoter; (iv) diluting and culturing the fused cells inseparate wells in a medium which will not support the unfused myelomacells; (v) evaluating the supernatant in each well containing ahybridoma for the presence of antibody to E rosette positive purified Tcells or human thymocytes; (vi) selecting and cloning a hybridomaproducing antibody which reacts with approximately 70% of normal humanthymocytes but not with normal human peripheral T cells, B cells, nullcells, or bone marrow cells; and (vii) recovering the antibody from thesupernatant above said clones.
 7. A monoclonal antibody which reactswith approximately 70% of normal human thymocytes but not with normalhuman peripheral T cells, B cells, null cells, or bone marrow cells,prepared by the method which comprises the steps of:(i) immunizing micewith purified human thymocytes; (ii) removing the spleens from said miceand making a suspension of the spleen cells; (iii) fusing said spleencells with mouse myeloma cells in the presence of a fusion promoter;(iv) diluting and culturing the fused cells in separate wells in amedium which will not support the unfused myeloma cells; (v) evaluatingthe supernatant in each well containing a hybridoma for the presence ofantibody to E rosette positive purified T cells or human thymocytes;(vi) selecting and cloning a hybridoma producing antibody which reactswith approximately 70% of normal human thymocytes but not with normalhuman peripheral T cells, B cells, null cells, or bone marrow cells;(vii) transferring said clones intraperitoneally into mice; and (viii)harvesting the malignant ascites or serum from said mice, which ascitesor serum contains the desired antibody.
 8. A method of preparingmonoclonal antibody which reacts with approximately 70% of normal humanthymocytes but not with normal human peripheral T cells, B cells, nullcells, or bone marrow cells, which comprises culturing the hybridomaATCC CRL 8020 in a suitable medium and recovering the antibody from thesupernatant above said hybridoma.
 9. The monoclonal antibody prepared bythe method of claim
 8. 10. A method of preparing monoclonal antibodywhich reacts with approximately 70% of normal human thymocytes but notwith normal human peripheral T cells, B cells, null cells, or bonemarrow cells, which comprises injecting into a mouse the hybridoma ATCCCRL 8020 and recovering the antibody from the malignant ascites or serumof said mouse.
 11. The monoclonal antibody prepared by the method ofclaim 10.